Controlling metabolic pathways in eukaryotic organisms is desirable for the purposes of producing novel traits therein or introducing novel traits into a particular cell, tissue or organ of said organism. Whilst recombinant DNA technology has provided significant progress in an understanding of the mechanisms regulating eukaryotic gene expression, much less progress has been made in the actual manipulation of gene expression to produce novel traits. Moreover, there are only limited means by which human intervention may lead to a modulation of the level of eukaryotic gene expression.
One approach to repressing, delaying or otherwise reducing gene expression utilise a mRNA molecule which is transcribed from the complementary strand of a nuclear gene to that which is normally transcribed and capable of being translated into a polypeptide. Although the precise mechanism involved in this approach is not established, it has been postulated that a double-stranded mRNA may form by base pairing between the complementary nucleotide sequences, to produce a complex which is translated at low efficiency and/or degraded by intracellular ribonuclease enzymes prior to being translated.
Alternatively, the expression of an endogenous gene in a cell, tissue or organ may be suppressed when one or more copies of said gene, or one or more copies of a substantially similar gene are introduced into the cell. Whilst the mechanism involved in this phenomenon has not been established and appears to be involve mechanistically heterogeneous processes. For example, this approach has been postulated to involve transcriptional repression, in which case somatically-heritable repressed states of chromatin are formed or alternatively, a post-transcriptional silencing wherein transcription initiation occurs normally but the RNA products of the co-suppressed genes are subsequently eliminated.
The efficiency of both of these approaches in targeting the expression of specific genes is very low and highly variable results are usually obtained. Inconsistent results are obtained using different regions of genes, for example 5′-untranslated regions, 3′-untranslated regions, coding regions or intron sequences to target gene expression. Accordingly, there currently exists no consensus as to the nature of genetic sequences which provide the most efficient means for repressing, delaying or otherwise reducing gene expression using existing technologies. Moreover, such a high degree of variation exists between generations such that it is not possible to predict the level of repression of a specific gene in the progeny of an organism in which gene expression was markedly modified.
Recently, Dorer and Henikoff (1994) demonstrated the silencing of tandemly repeated gene copies in the Drosophila genome and the transcriptional repression of dispersed Drosophila Adh genes by Polycomb genes (i.e. the Pc-G system; Pal-Bhadra et al, 1997). However, such silencing of tandemly repeated gene copies is of little utility in an attempt to manipulate gene expression in an animal cell by recombinant means, wherein the sequences capable of targeting the expression of a particular gene are introduced at dispersed locations in the genome, absent the combination of this approach with gene-targeting technology. Whilst theoretically possible, such combinations would be expected to work at only low-efficiency, based upon the low efficiency of gene-targeting approaches used in isolation and further, would require complicated vector systems. Additionally, the utilisation of transcriptional repression, such as the Drosophila Pc-G system, would appear to require some knowledge of the regulatory mechanisms capable of modulating the expression of any specific target gene and, as a consequence, would be difficult to implement in practice as a general technology for repressing, delaying or reducing gene expression in animal cells.
The poor understanding of the mechanisms involved in these phenomena has meant that there have been few improvements in technologies for modulating the level of gene expression, in particular technologies for delaying, repressing or otherwise reducing the expression of specific genes using recombinant DNA technology.
Furthermore, as a consequence of the unpredictability of these approaches, there is currently no commercially-viable means for modulating the level of expression of a specific gene in a eukaryotic or prokaryotic organism.
Thus, there exists a need for improved methods of modulating gene expression, in particular repressing, delaying or otherwise reducing gene expression in animal cells for the purpose of introducing novel phenotypic traits thereto. In particular, these methods should provide general means for phenotypic modification, without the necessary for performing concomitant gene-targeting approaches.